Study Of The Mechanisms Of The ATP-dependent Protein ClpB In Stress Tolerance And Virulence Of Streptococcus Agalactiae

Streptococcus agalactiae is a Gram-positive bacterium with a wide host range,capable of infecting humans and a wide range of animals,including tilapia.S.agalactiae,regarded as the major bacterial pathogen of farmed Nile tilapia(Oreochromis niloticus),causes high mortality and huge economic losses.Bacterial ClpB,an ATP-dependent disaggregase that belongs to the Hsp100/Clp family,disaggregates stress-denatured proteins and promotes bacterial survival under adverse environmental conditions.Since there is currently a knowledge vacuum about ClpB in S.agalactiae,this study constructed a clpB deletion mutant and first looked into how ClpB works in S.agalactiae,as follows.First,we reported a ClpB homolog in S.agalactiae and constructed a clpB deletion mutant(?clpB)to explore the function.The ?clpB exhibits no defective growth or morphology under 28℃ conditions,but had a defective tolerance against heat and acidic stress: its survival rates decreased by 33.58% and 89.41% respectively.It indicates that ClpB contributes to mediate tolerance to stressful conditions,in particular heat and low p H.Second,the intracellular survival of ΔclpB in RAW264.7 cells decreased by 23.78%,and the adherence to tilapia brain cells diminished by 10.91%.Similarly,ΔclpB showed increased sensitivity to tilapia head kidney leukocytes and whole blood killing.In comparison to ΔclpB,HN016 survived in tilapia head kidney leukocytes at approximately2.92 times and proliferated at approximately 1.68 times in whole blood.In the tilapia infection assay,ΔclpB showed a 40% reduction in mortality and a significant reduction in bacterial loads.The results of the virulence assay demonstrate that ClpB mediates intracellular survival and host colonization and plays an important role in the pathogenic process of S.agalactiae.Further,reduced virulence of the ?clpB was explored through transcriptome analysis,identifying 155 differentially expressed genes(DEGs)that were involved in the bacterial stress response.Gene Ontology enrichment and Kyoto Encyclopedia of genes and genomes enrichment analyses were mainly annotated to pathways related to metabolism pathways,suggesting that ClpB indirectly regulates the virulence of S.agalactiae by regulating stress responses and metabolic pathways.Taken together,this study firstly demonstrated that ClpB,as a molecular chaperone,plays critical roles in heat and acidic stress resistance and virulence of S.agalactiae.This finding enriches the understanding of the function of ClpB homolog in gram-positive bacteria and the survival strategy of S.agalactiae against immune cleaning during the infection process,which provides a scientific basis for further research of pathogenesis in S.agalactiae and provides new targets for novel antimicrobial therapies in combating bacterial infections.